Bottom Line:
We show that non-MHC genes from the NOD strain cause a failure to delete these high avidity autoreactive T cells during their development in the thymus, with subsequent spontaneous breakdown of CD4 cell tolerance to the islet antigen, formation of intra-islet germinal centers, and high titre immunoglobulin G1 autoantibody production.In mixed bone marrow chimeric animals, defective thymic deletion was intrinsic to T cells carrying diabetes susceptibility genes.These results demonstrate a primary failure to censor forbidden clones of self-reactive T cells in inherited susceptibility to organ-specific autoimmune disease, and highlight the importance of thymic mechanisms of tolerance in organ-specific tolerance.

Affiliation: Australian Cancer Research Foundation Genetics Lab, Medical Genome Centre, John Curtin School of Medical Research, Australian National University, Canberra ACT 2601, Australia.

ABSTRACTType 1 diabetes and other organ-specific autoimmune diseases often cluster together in human families and in congenic strains of NOD (nonobese diabetic) mice, but the inherited immunoregulatory defects responsible for these diseases are unknown. Here we track the fate of high avidity CD4 T cells recognizing a self-antigen expressed in pancreatic islet beta cells using a transgenic mouse model. T cells of identical specificity, recognizing a dominant peptide from the same islet antigen and major histocompatibility complex (MHC)-presenting molecule, were followed on autoimmune susceptible and resistant genetic backgrounds. We show that non-MHC genes from the NOD strain cause a failure to delete these high avidity autoreactive T cells during their development in the thymus, with subsequent spontaneous breakdown of CD4 cell tolerance to the islet antigen, formation of intra-islet germinal centers, and high titre immunoglobulin G1 autoantibody production. In mixed bone marrow chimeric animals, defective thymic deletion was intrinsic to T cells carrying diabetes susceptibility genes. These results demonstrate a primary failure to censor forbidden clones of self-reactive T cells in inherited susceptibility to organ-specific autoimmune disease, and highlight the importance of thymic mechanisms of tolerance in organ-specific tolerance.

fig3: Failure to downregulate TCR expression in splenocytes of NODk animals. (a) Spleen cells from nondiabetic female mice of various genotypes were stained with antibodies to CD4, CD3, Vβ8, and 1G12 clonotype. Histograms show profiles of CD4-positive cells from mice of the indicated genotypes. (b) Vα3 expression on CD4+Vβ8+ spleen cells from B10k mice of the indicated genotypes. (c) Staining for an endogenous TCR-α chain, Vα2, on CD4+ spleen cells. The percentage of CD4+ cells that are Vα2+ is indicated.

Mentions:
Receptor downregulation is an important means of diminishing antigen reactivity, and almost all CD4+ T cells in the periphery of TCR/insHEL B10k mice display half the surface density of CD3 and Vβ8 that are found on TCR transgenic mice without the negative-selecting antigen (Fig. 3 A and Fig. 4). Most of these T cells carry the transgenic Vα3 chain on their surface, but at 10-fold reduced levels compared with TCR cells (Fig. 3 B) so that they are difficult to detect by staining with the clonotypic 1G12 antibody (Fig. 3 A). Since transgenic Vβ8 chains on these cells are only reduced by 50%, the remaining receptors must contain a second TCR-α chain. This conclusion is supported by the fact that Vα2 chains are expressed by a fraction of these cells, and by the finding that the level of Vα2 displayed on their surface is about half that found on nontransgenic mice (Fig. 3 C). By contrast, very little decrease in CD3 or Vβ8 occurred on peripheral CD4 T cells from NODk TCR/insHEL mice, and a large fraction still express high surface densities of clonotypic receptor 1G12 (Fig. 3 A). Thus, insHEL caused a dramatic reduction in the avidity of peripheral T cells reacting with HEL on the B10k background, but induced little change compared with TCR-transgenic animals without HEL on the NODk background.

fig3: Failure to downregulate TCR expression in splenocytes of NODk animals. (a) Spleen cells from nondiabetic female mice of various genotypes were stained with antibodies to CD4, CD3, Vβ8, and 1G12 clonotype. Histograms show profiles of CD4-positive cells from mice of the indicated genotypes. (b) Vα3 expression on CD4+Vβ8+ spleen cells from B10k mice of the indicated genotypes. (c) Staining for an endogenous TCR-α chain, Vα2, on CD4+ spleen cells. The percentage of CD4+ cells that are Vα2+ is indicated.

Mentions:
Receptor downregulation is an important means of diminishing antigen reactivity, and almost all CD4+ T cells in the periphery of TCR/insHEL B10k mice display half the surface density of CD3 and Vβ8 that are found on TCR transgenic mice without the negative-selecting antigen (Fig. 3 A and Fig. 4). Most of these T cells carry the transgenic Vα3 chain on their surface, but at 10-fold reduced levels compared with TCR cells (Fig. 3 B) so that they are difficult to detect by staining with the clonotypic 1G12 antibody (Fig. 3 A). Since transgenic Vβ8 chains on these cells are only reduced by 50%, the remaining receptors must contain a second TCR-α chain. This conclusion is supported by the fact that Vα2 chains are expressed by a fraction of these cells, and by the finding that the level of Vα2 displayed on their surface is about half that found on nontransgenic mice (Fig. 3 C). By contrast, very little decrease in CD3 or Vβ8 occurred on peripheral CD4 T cells from NODk TCR/insHEL mice, and a large fraction still express high surface densities of clonotypic receptor 1G12 (Fig. 3 A). Thus, insHEL caused a dramatic reduction in the avidity of peripheral T cells reacting with HEL on the B10k background, but induced little change compared with TCR-transgenic animals without HEL on the NODk background.

Bottom Line:
We show that non-MHC genes from the NOD strain cause a failure to delete these high avidity autoreactive T cells during their development in the thymus, with subsequent spontaneous breakdown of CD4 cell tolerance to the islet antigen, formation of intra-islet germinal centers, and high titre immunoglobulin G1 autoantibody production.In mixed bone marrow chimeric animals, defective thymic deletion was intrinsic to T cells carrying diabetes susceptibility genes.These results demonstrate a primary failure to censor forbidden clones of self-reactive T cells in inherited susceptibility to organ-specific autoimmune disease, and highlight the importance of thymic mechanisms of tolerance in organ-specific tolerance.

Affiliation:
Australian Cancer Research Foundation Genetics Lab, Medical Genome Centre, John Curtin School of Medical Research, Australian National University, Canberra ACT 2601, Australia.

ABSTRACTType 1 diabetes and other organ-specific autoimmune diseases often cluster together in human families and in congenic strains of NOD (nonobese diabetic) mice, but the inherited immunoregulatory defects responsible for these diseases are unknown. Here we track the fate of high avidity CD4 T cells recognizing a self-antigen expressed in pancreatic islet beta cells using a transgenic mouse model. T cells of identical specificity, recognizing a dominant peptide from the same islet antigen and major histocompatibility complex (MHC)-presenting molecule, were followed on autoimmune susceptible and resistant genetic backgrounds. We show that non-MHC genes from the NOD strain cause a failure to delete these high avidity autoreactive T cells during their development in the thymus, with subsequent spontaneous breakdown of CD4 cell tolerance to the islet antigen, formation of intra-islet germinal centers, and high titre immunoglobulin G1 autoantibody production. In mixed bone marrow chimeric animals, defective thymic deletion was intrinsic to T cells carrying diabetes susceptibility genes. These results demonstrate a primary failure to censor forbidden clones of self-reactive T cells in inherited susceptibility to organ-specific autoimmune disease, and highlight the importance of thymic mechanisms of tolerance in organ-specific tolerance.